The cell cycle for growing cells can be divided into two periods: (1) the cell division period, when the cell divides and separates, with each daughter cell receiving identical copies of the DNA; and (2) the period of growth, known as the interphase period. For the cell cycle of eucaryotes, the cell division period is labeled the M (mitotic) period. The interphase period in eucaryotes is further divided into three successive phases: G1 (gap 1) phase, which directly follows the M period; S (DNA synthetic) phase, which follows G1; and G2 (gap 2) phase, which follows the S phase, and immediately precedes the M period. During the two gap phases no net change in DNA occurs, though damaged DNA may be repaired. On the other hand, throughout the interphase period there is continued cellular growth and continued synthesis of other cellular components. Towards the end of the G1 phase, the cell passes a restrictive (R) point and becomes committed to duplicate its DNA. At this point, the cell is also committed to divide. During the S phase, the cell replicates DNA. The net result is that during the G2 phase, the cell contains two copies of all of the DNA present in the G1 phase. During the subsequent M period, the cells divide with each daughter cell receiving identical copies of the DNA. Each daughter cell starts the next round of the growth cycle by entering the G1 phase.
The G1 phase represents the interval in which cells respond maximally to extracellular signals, including mitogens, anti-proliferative factors, matrix adhesive substances, and intercellular contacts. Passage through the R point late in G1 phase defines the time at which cells lose their dependency on mitogenic growth factors for their subsequent passage through the cycle and, conversely, become insensitive to anti-proliferative signals induced by compounds such as transforming growth factor, cyclic AMP analogs, and rapamycin. Once past the R point, cells become committed to duplicating their DNA and to undergoing mitosis, as noted above, and the programs governing these processes are largely cell autonomous.
In mammalian cells, a molecular event that temporally coincides with passage through the R point is the phosphorylation of the retinoblastoma protein (Rb). In its hypophosphorylated state, Rb prevents the cell from exiting the G1 phase by combining with transcription factors such as E2F to actively repress transcription from promoters containing E2F binding sites. However, hyperphosphlorylation of Rb late in G1 phase prevents its interaction with E2F, thus allowing E2F to activate transcription of the same target genes. As many E2F-regulated genes encode proteins that are essential for DNA synthesis, Rb phosphorylation at the R point helps convert cells to a pre-replicative state that anticipates the actual G1/S transition by several hours.
Regulation of the human cell cycle requires the periodic formation, activation, and inactivation of protein kinase complexes that consist of a regulatory "cyclin" subunit and a catalytic "cyclin dependent kinase" (CDK) subunit. Cell cycle-dependent fluctuations in the levels of many of the cyclin proteins contribute to the activation of these protein kinase complexes. For example, cyclin B participates in the regulation of the G2/M transition by its association with its catalytic subunit, p34 .sup.cdc2, whereas cyclin A, in complexes with both p34.sup.cdc2 and CDK2, is essential for the completion of S-phase and entry into G2-phase. Complexes formed between the D-type cyclins and either CDK4 or CDK6 integrate growth factor signals and the cell cycle, allowing cells to progress through G1-phase. This particular cell cycle pathway is specifically altered during tumorigenesis, presumably due to its role in responses to mitogenic stimulation. Alterations have been identified in many components of this pathway, including the D-type cyclins, CDKs, and cyclin dependent kinase inhibitors (CKIs). Another G1-phase cyclin, cyclin E, in conjunction with its catalytic subunit CDK2, appears to be essential for progression from G1-phase into S-phase and the initiation of DNA replication. Cyclin E and CDK2 do not appear to be directly targeted during tumorigenesis, quite possibly due to their essential nature. [See generally, Sherr, Cell 73:1059-1065 (1993), and Sherr, Cell 79:551-555 (1994)].
A class of novel polypeptides that are collectively known as CDK inhibitors (CKIs) can negatively regulate cyclin/CDK activity by associating with these complexes. These so-called "cell cycle brakes" act to inhibit cyclin/CDK complexes by binding specifically to either CDK, (i.e.,the INK4s, see below) or the cyclin/CDK complexes (i.e., KIP1/CIP1, see below). CKI activity and levels are cell cycle regulated allowing these proteins to function as inhibitors of their cognate cyclin/CDK complexes for very limited periods during the cell cycle,
There are two types of CKIs that have been identified, the INK4s, and the CIP/KIPs [Sherr & Roberts, Genies Devel. 9:1149-1163 (1995)]. The INK4 family of inhibitors comprises four members, p16.sup.INK4a, p15.sup.INK4b, p18.sup.INK4c and p19.sup.INK4d, which specifically bind to and inhibit G1-specific CDK4 and CDK6, and thereby prevent phosphorylation of the retinoblastoma (Rb) protein and S phase entry. The CIP/KIP family of inhibitors includes p21.sup.CIP1, p27.sup.KIP1, and p57.sup.KIP2, and unlike the INK4 proteins, can inhibit all cyclin/CDK complexes [Harper & Elledge, Curr. Opin. Genet. Dev. 6:56-64 (1996)]. This apparent redundancy in CDK complex inhibitors has been explained as a method for organisms to govern transitions through the R point in different cell types responding to a plethora of distinct extracellular signals.
Despite their apparent biochemical redundancy, the CKIs are differentially expressed during mammalian (e.g., mouse) development and in adult tissues, showing some overlapping expression patterns. Deletion of a single CKI or in combination with another in the mouse leads to specific phenotypes, but in spite of their reported expression in the central nervous system (CNS) none give rise to a reported phenotype in the brain. For example, mice lacking p57.sup.KIP2, die soon after birth and display developmental defects mimicking those observed in human patients with Beckwith-Weideman syndrome [Zhang et al., Nature 387:151-158 (1997); Yan et al., Genes and Development 11: 973-983 (1997)]. Mice lacking p27.sup.KIP1 develop organomegaly, neurological conditions i.e., display retinal dysplasia, female sterility, and benign pituitary adenomas [Fero et al., Cell 85:733-744 (1996); Kiyokawa et al., Cell 85:721-732 (1996); Nakayama et al., Cell 85:707-720 (1996)]. However, mice deleted for the other CKIs examined heretofore, do not display apparent phenotypes. Mice lacking p21.sup.CIP1 [Deng et al., Cell 82:675-684 (1995)] are developmentally normal and do not develop spontaneous tumors. Mice lacking p18.sup.INK4c develop gigantism, widespread organomegaly and pituitary adenomas by 10 months of age [Franklin et al., Genes and Development 12:2899-2911 (1998)], whereas mice lacking both p18.sup.INK4c and p27.sup.KIP1 develop pituitary adenocarcinomas with an accelerated onset from what is seen in the p18.sup.INK4c -double null animals (i.e., animals lacking both p18.sup.INK4c alleles) [Franklin et al., Genes and Development 12:2899-2911 (1998)]. Similarly, deletion of p27.sup.KIP1 and p57.sup.KIP2 leads to aberrant proliferation of these cells due to inhibition of cell cycle exit and differentiation in these tissues. In wild type mice, both p27.sup.KIP1 and p57.sup.KIP2 are expressed in the lens fiber cells and in placental trophoblasts, [Zhang et al., Genes and Development 12:3162-3167 (1998)].
Development of the central nervous system (CNS) requires proliferation of neuronal and glial cell precursors followed by their subsequent differentiation in a highly coordinated manner. However, despite the obvious need for replacement of neuronal cells in cases of neural injuries and diseases, to date, no such cell source is available. Therefore, there is presently a need for methodology that can provide an avenue for stimulating the growth of neuronal populations. Furthermore, there are relatively few if any animal models for diseases that include symptoms such as bradykinesia. Therefore, there is a need for animal models for this and other neurological maladies and disorders.
The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application.